Column Chromatography: Can you Separate the Dyes in Grape Soda Using Space Sand™?
Abstract What color is grape soda? If you pour it into a clear glass you can easily see it is purple, but that is usually not its natural color. Manufacturers add red and blue dye to the soda. The dyes mix together and you get purple soda. What if you wanted to un-mix the dyes, could you? Yes! In a chemistry laboratory, using a technique called column chromatography, you could separate the two dyes again. But what about at home, can you use low-tech supplies to do the same thing? In this science project you will try to do just that using some interesting materials including a toy/craft product called Space Sand. Objective Investigate whether a homemade column chromatography setup can be used to separate and isolate the different food colorings that are in grape soda. Materials and Equipment *Plastic syringes with Luer-Lock, 30 mL, without needles (4); available online at Amazon.com *Space Sand (24 mL, or about 2 Tbsp.); available at some toy stores or online at Amazon.com. White sand is recommended so that dyes from the sand do not potentially interfere with results. *Scissors *Optional: Funnel with a tip that fits into the top of the syringe once the plunger is pulled out *Clear plastic cups or drinking glasses, at least 5-oz. (8) *Permanent marker or sticky note and pen or pencil *Isopropyl alcohol (150 mL) *Distilled water (90 mL); available at most grocery stores *Grape soda (30 mL) *Lab notebook *Optional: Digital camera to take pictures of your experiment for your project display board Experimental Procedure Using Column Chromatography on Grape Soda #In your lab notebook, make data tables like Table 1 and Table 2, below. You will be recording your observations in these data tables. ##Remember that the eluate is the liquid that comes out the bottom of the column, and that each different cup in which the eluate is collected is referred to as a different fraction. Table 1. In your lab notebook, make a data table like this one to record your eluate color results in. Table 2. In your lab notebook, make a data table like this one to record your column sand observations in.#Get out three plastic cups or drinking glasses. Pour some 70% isopropyl alcohol into one, distilled water into another, and grape soda into the last cup. ##Specifically, you will need at least 150 mL of the isopropyl alcohol, 90 mL distilled water, and 30 mL grape soda. But you do not need to carefully measure out these quantities now because you will measure them later, as you use them. ##You could label the cups (with either a permanent marker if they are plastic or with a sticky note and pen or pencil if they are glass) or just keep them near the containers they came from so you know what is in each cup. #Get out five more plastic cups and label them Waste, 1st Fraction, 2nd Fraction, 3rd Fraction, and 4th Fraction. It will be easiest to arrange them in a line in front of you, going left to right. #Remove one of the 30 milliliter (mL) syringes from its packaging. Use a permanent marker to label the syringe "A" on the side somewhere. #Pull the syringe's plunger all the way out and fill the syringe with 8 mL of packed Space Sand, as shown in Figure 5, below. ##''Note:'' Some sand will inevitably come out the bottom of the syringe. If the syringe came with a cap, as shown in Figure 5, you can keep the cap on to help prevent sand from falling out of the syringe while you pack it. ##''Tip:'' To fill the syringe with the sand, it might be easiest to use scissors to cut off a small corner of the sand's bag and slowly pour it out from that corner, and into the syringe. Alternatively you may use a funnel. ##As you fill the syringe with the sand, repeatedly tap the syringe on a hard surface so that the sand becomes compacted. If needed, use the syringe plunger to push sand down and off the inner sides of the syringe. ##When you think you are done, make sure that the sand is not less than 8 mL after you have tapped the column several times. ##Keep in mind that it is much better to have a little too much sand (such as 8–9 mL) in the syringe than too little sand (less than 8 mL). Why do you think this is? ##When it is ready, carefully set the sand-packed syringe aside for now. #First equilibrate your column with the mobile phase and some distilled water. This will prepare it for processing the grape soda. ##If it is not already, pull the plunger out of the sand-packed syringe, which you labeled "A," and carefully set the syringe in the cup labeled Waste. ##Get out a second 30 mL syringe. Use a permanent marker to label this syringe "B." Use syringe B to suck up 30 mL of 70% isopropyl alcohol from its cup. ##Hold syringe A (the sand-packed syringe) over the waste cup. Then, in your other hand, hold syringe B over syringe A and slowly push out the alcohol from syringe B into syringe A. See Figure 5, below, to see how the syringes should be arranged. ###Let the alcohol drip out of syringe A and into the waste cup below. ###To speed up the process, you can put syringe A's plunger back into the syringe and push out the remaining alcohol once you have squeezed it all out of syringe B. ###''Note:'' When doing real column chromatography, it is often important to never let the column run dry. In other words, once a column is damp, it should stay damp. However, in this science project it is okay to push the liquid out of the sand if the sand will be dry for just a minute or two. While, in general, it is better to let the sand stay a little damp, there should be no remaining liquid above the level of the sand in the syringe. ###Do not be alarmed if you see some sand come out. It is normal to lose a little sand during this process. ##Once all the alcohol has dripped out, pull the plunger out of syringe A, but continue to hold it over the waste cup. ##Take syringe B and use it to suck up 30 mL of distilled water. ##Repeat step c. with the distilled-water-filled syringe (instead of the alcohol-filled syringe). ##Your column has now been equilibrated and it is ready to use with the grape soda. Record the color of the equilibrated sand in the data table (the one similar to Table 2, above) in your lab notebook. #Now add a sample of grape soda to your equilibrated column. ##Pull the plunger out of syringe A and carefully set the syringe in the cup labeled 1st Fraction. ##Take syringe B and use it to slowly suck up 10 mL of grape soda. Note: Be sure you slowly suck it up to prevent bubbles from accumulating in the syringe. ##Hold syringe A over the 1st Fraction cup. Then, in your other hand, hold syringe B over syringe A and slowly push the grape soda out of syringe B and into syringe A, as you did to equilibrate the column. ###You can use syringe A's plunger to speed up this process, but remember not to leave the sand dry for more than a minute or two. ##What is the color of the eluate that comes out in this fraction? What is the color of the sand in the syringe? Write your observations in the data tables in your lab notebook. #Next add your 70% isopropyl alcohol mobile phase to the column. ##Pull the plunger out of syringe A and carefully set the syringe in the cup labeled 2nd Fraction. ##Take syringe B and use it to suck up 10 mL of the 70% isopropyl alcohol. ##Hold syringe A over the 2nd Fraction cup. Then, in your other hand, hold syringe B over syringe A and slowly push out the alcohol into syringe A, as you did before. ##''Important:'' Be sure you closely watch the liquid drip out of syringe A, and as soon as the drops look like they are changing color, move syringe A so that it is over the 3rd fraction cup. Then let the remaining liquid drip, or be pushed out, into this cup. ###You can use syringe A's plunger to speed up this process, but remember not to leave the sand dry for more than a minute or two. ##What is the color of the eluate that comes out in the 2nd and 3rd Fraction cups? Record all of your observations in the data tables in your lab notebook. #Lastly, add some more of your 70% isopropyl alcohol to the column. ##Pull the plunger out of syringe A and carefully set the syringe in the cup labeled 4th Fraction. ##Take syringe B and use it to suck up 10 mL more of 70% isopropyl alcohol. ##Hold syringe A over the 4th Fraction cup. Then, in your other hand, hold syringe B over syringe A and slowly push out the alcohol into syringe A, as you did before. ###If you use syringe A's plunger to speed up this process, be sure to leave the sand damp, since you do not want it to be dry while you take time to prepare for the next trial. ##What is the color of the eluate that comes out in the 4th Fraction cup? What is the color of the column's sand? Write your observations in the data tables in your lab notebook. ##Do not discard your results from each trial; you will use them for comparison when you analyze your results. #If you want to, you can take pictures of your eluates in the different cups. You should include a sample of the original grape soda in a cup for comparison. You may want to take pictures using a white background, such as a sheet of blank paper, and include labels. You could include these pictures on yourScience Fair Project Display Board. #Repeat steps 3–10 two more times. Be sure to record your observations in the data tables in your lab notebooks, in the rows for trials 2 and 3. ##For syringe "A," use a new syringe, but you can use the same syringe as before for syringe "B." ##Repeat step 2 if you need more liquid in those cups. Analyzing Your Results #When you have completed the column chromatography, compare the liquids in the cups and the results you recorded in the data tables in your lab notebook. ##Are the colors of the eluates different? For example, are the fractions collected from when you added the grape soda different from the fractions collected when you added the 70% isopropyl alcohol? If so, how are they different? Why do you think this is? ##Were your results consistent between your different trials, or was there some variability? ##If the eluates from the different fractions are different colors, what do you think this has to do with how hydrophobic the different food colorings in the grape soda are? Hint: Try re-reading the Introduction to figure this out, thinking about how 70% isopropyl alcohol might interact with the different food colorings in the grape soda. ##Overall, was your homemade column chromatography setup successful at separating the food colorings in the grape soda? If it worked, can you explain how? Variations *Many different artificial beverages, dry beverage mixes, and other foods contain different food colorings. Use your column chromatography setup to separate food coloring mixtures in other beverages or food. Do you need to change the stationary and/or mobile phase(s) for it to work? *In this science project, you created a homemade column chromatography setup that may have worked well, but can you make it work even better? For example, could changing the amount of grape soda, type of mobile phase, number of collection cups used, or amount of sand in the column improve your results? (Important: If you use a different mobile phase, be sure to take the proper safety precautions for using any of the chemicals.) Or, if it was a problem, could you prevent the sand from leaking out of the syringe, such as by using a tiny bit of cotton ball in the bottom of the syringe or by wrapping it with a nylon stocking? What about trying different concentrations of your mobile phase? You could check out the resources in the Bibliography in theBackground for more information on column chromatography and to get ideas of how to improve your setup. *Make a column chromatography setup that is even easier for people to make at home. For example, you probably had to order syringes to do this science project. Could you replace them with something that is more common, such as plastic water bottles that have their bottoms cut off and are flipped upside down? If you do this, how can you prevent the sand from leaking out? Since the bottle is probably larger than the syringe, will you need to increase the amount of materials (e.g., sand, grape soda, isopropyl alcohol, etc.) that you use? Can you let the mobile phase flow out just by using gravity? There can be a lot of challenges, but making a technique like this available to more people can be very rewarding! *You can make your own hydrophobic sand at home and then compare how it works in your homemade column chromatography setup to how the Space Sand worked. To make hydrophobic sand, make a thin one- to two-grain-thick layer of sand on a cookie sheet and spray the sand with a spray-on water repellent. Be sure to work in a well-ventilated area, follow all safety precautions, and use a cookie sheet that can be discarded afterwards (you should not use it with food). You will want to repeat this process several times to thoroughly coat the sand: Mix the sand after spraying it to spread around the spray, let the sand dry, turn the sand over and spray it again. Let the sand completely dry before testing it with a drop of water to see if it is hydrophobic (if it is, it should look similar to Figure 4 in the Introduction). Does it work as well, or better than, the Space Sand? *For other science projects on chromatography see: **Paper Chromatography: Is Black Ink Really Black? **Candy Chromatography: What Makes Those Colors? **Paper Chromatography: Advanced Version 1 **Paper Chromatography: Advanced Version 2 **Reveal the Red: Exploring the Chemistry of Red Flower Pigments **Sizing It Up! How Scientists Separate Proteins *For other science projects on food coloring dyes see: **Candy Chromatography: What Makes Those Colors? **Forensic Science: Building Your Own Tool for Identifying DNA **What Makes Candies So Colorful? Investigate How Gel Electrophoresis Unlocks the Color Code! Photos Category:Biotechnology Experiments